结合反相高效液相色谱和酶联免疫吸附法测定昆虫血淋巴中的蜕皮甾醇激素
Detection of ecdysteroids in insect hemolymph by a combination ofreverse-phase high-performance lipid chromatography and anenzyme linked immunosorbent assay
叶 滨1, 2** 李 康2 李 胜2*** 李 恺1***
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DOI：10.7679/j.issn.2095-1353.2016.056
作者单位：1. 华东师范大学生命科学学院，上海 200241；2. 中国科学院上海生命科学研究院植物生理生态研究所，上海 200032
中文关键词：家蚕,蜕皮甾醇激素，蜕皮酮，20羟基蜕皮酮，反相HPLC，酶联免疫吸附法
英文关键词：Bombyx, hemolymph, ecdysteroid, ecdyosne, 20-hydroxyecdysone, RHPLC, ELISA
中文摘要：
【目的】 昆虫体内的蜕皮酮和20羟基蜕皮酮（20E）是两种主要的蜕皮甾醇激素，其中蜕皮酮是20E的前体，而20E是有活性的蜕皮甾醇激素。本研究旨在建立一种测定昆虫血淋巴中蜕皮激素高效、稳定、准确的方法。【方法】 采集家蚕血淋巴，抽提总蜕皮甾醇激素，利用反相高效液相色谱法（反相HPLC）分离并收集蜕皮酮与20E，进而利用酶联免疫吸附法（ELISA）测定家蚕血淋巴的总蜕皮甾醇激素、蜕皮酮和20E各自的滴度。【结果】 计算出总蜕皮甾醇激素中蜕皮酮与20E的比例，推算出蜕皮酮合成和分泌、以及蜕皮酮转化为20E的能力。【结论】 该方法高效、稳定、准确，可广泛应用于昆虫蜕皮甾醇激素滴度的测定。
英文摘要：
[Objectives] To develop a highly efficient, stable, and accurate, method for the determination of two main ecdysteroids in insect hemolymph, namely ecdysone and 20-hydroxyecdysone (20E). Ecdysone is the immediate precursor of 20E, while 20E is the activated form of insect ecdysteroids, or molting hormones. [Methods] Ecdysone and 20E collected in Bombyx hemolymph were separated by reverse-phase, high-performance, liquid chromatography (RHPLC), followed by an enzyme linked immunosorbent assay (ELISA), to determine their respective titers. [Results] The ecdysone and 20E titer in Bombyx hemolymph was determined, and the relative ratio of ecdysone to 20E obtained. The biosynthesis and secretion rates of ecdysone, as well as the conversion rate of ecdysone to 20E, were calculated. [Conclusion] The new method is highly efficient, stable, and accurate, and can be conveniently applied to determine the titers of ecdysteroids in insects.